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Chemoprevention of bilirubin encephalopathy with a nanoceutical agent

Pediatric Research (2022)Cite this article

Abstract

Background

Targeted rapid degradation of bilirubin has the potential to thwart incipient bilirubin encephalopathy. We investigated a novel spinel-structured citrate-functionalized trimanganese tetroxide nanoparticle (C-Mn3O4 NP, the nanodrug) to degrade both systemic and neural bilirubin loads.

Method

Severe neonatal unconjugated hyperbilirubinemia (SNH) was induced in neonatal C57BL/6j mice model with phenylhydrazine (PHz) intoxication. Efficiency of the nanodrug on both in vivo bilirubin degradation and amelioration of bilirubin encephalopathy and associated neurobehavioral sequelae were evaluated.

Results

Single oral dose (0.25 mg kg−1 bodyweight) of the nanodrug reduced both total serum bilirubin (TSB) and unconjugated bilirubin (UCB) in SNH rodents. Significant (p < 0.0001) UCB and TSB-degradation rates were reported within 4–8 h at 1.84 ± 0.26 and 2.19 ± 0.31 mg dL−1 h−1, respectively. Neural bilirubin load was decreased by 5.6 nmol g−1 (p = 0.0002) along with improved measures of neurobehavior, neuromotor movements, learning, and memory. Histopathological studies confirm that the nanodrug prevented neural cell reduction in Purkinje and substantia nigra regions, eosinophilic neurons, spongiosis, and cell shrinkage in SNH brain parenchyma. Brain oxidative status was maintained in nanodrug-treated SNH cohort. Pharmacokinetic data corroborated the bilirubin degradation rate with plasma nanodrug concentrations.

Conclusion

This study demonstrates the in vivo capacity of this novel nanodrug to reduce systemic and neural bilirubin load and reverse bilirubin-induced neurotoxicity. Further compilation of a drug-safety-dossier is warranted to translate this novel therapeutic chemopreventive approach to clinical settings.

Impact

  • None of the current pharmacotherapeutics treat severe neonatal hyperbilirubinemia (SNH) to prevent risks of neurotoxicity.

  • In this preclinical study, a newly investigated nano-formulation, citrate-functionalized Mn3O4 nanoparticles (C-Mn3O4 NPs), exhibits bilirubin reduction properties in rodents.

  • Chemopreventive properties of this nano-formulation demonstrate an efficacious, efficient agent that appears to be safe in these early studies.

  • Translation of C-Mn3O4 NPs to prospective preclinical and clinical trials in appropriate in vivo models should be explored as a potential novel pharmacotherapy for SNH.

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Fig. 1: In vivo bilirubin degradation efficacy of C-Mn3O4 NPs nanodrug in SNH model (postnatal age 10 days).
Fig. 2: Effect of the nanodrug on prevention of SNH-associated neurotoxicity.
Fig. 3: Effect of the nanodrug on SNH-associated brain (cerebellar) oxidative distress.
Fig. 4: Effect of the nanodrug on SNH-associated neuromotor function.
Fig. 5: “Long-term” effects in learning and memory.
Fig. 6: Pharmacokinetics and pharmacodynamics of the nanodrug.

Data availability

All data that support the findings of this study are available within the published article. Study-related additional data will be available from S.K.P. upon legitimate request.

References

  1. Bhutani, V. K. & Stevenson, D. K. The need for technologies to prevent bilirubin-induced neurologic dysfunction syndrome. Semin. Perinatol. 35, 97–100 (2011).

    Article  PubMed  Google Scholar 

  2. Slusher, T. M., Zipursky, A. & Bhutani, V. K. A global need for affordable neonatal jaundice technologies. Semin. Perinatol. 35, 185–191 (2011).

    Article  PubMed  Google Scholar 

  3. Bhutani, V. K. et al. Clinical trial of tin mesoporphyrin to prevent neonatal hyperbilirubinemia. J. Perinatol. 36, 533–539 (2016).

    Article  CAS  PubMed  Google Scholar 

  4. Qattea, I., Farghaly, M. A. A., Elgendy, M., Mohamed, M. A. & Aly, H. Neonatal hyperbilirubinemia and bilirubin neurotoxicity in hospitalized neonates: analysis of the US database. Pediatr. Res. (2021).

  5. Olusanya, B. O., Teeple, S. & Kassebaum, N. J. The contribution of neonatal jaundice to global child mortality: findings from the GBD 2016 Study. Pediatrics 141, e20171471 (2018).

    Article  PubMed  Google Scholar 

  6. Fujiwara, R., Nguyen, N., Chen, S. & Tukey, R. H. Developmental hyperbilirubinemia and CNS toxicity in mice humanized with the UDP glucuronosyltransferase 1 (UGT1) locus. Proc. Natl Acad. Sci. USA 107, 5024 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Bhutani, V. K. et al. Neonatal hyperbilirubinemia and rhesus disease of the newborn: incidence and impairment estimates for 2010 at regional and global levels. Pediatr. Res. 74, 86–100 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Dandona, R. et al. Subnational mapping of under-5 and neonatal mortality trends in India: The Global Burden of Disease Study 2000–17. Lancet 395, 1640–1658 (2020).

    Article  Google Scholar 

  9. Valaes, T., Petmezaki, S., Henschke, C., Drummond, G. S. & Kappas, A. Control of jaundice in preterm newborns by an inhibitor of bilirubin production: studies with tin-mesoporphyrin. Pediatrics 93, 1–11 (1994).

    Article  CAS  PubMed  Google Scholar 

  10. Wong, R. J., Bhutani, V. K., Vreman, H. J. & Stevenson, D. K. Pharmacology review: tin mesoporphyrin for the prevention of severe neonatal hyperbilirubinemia. NeoReviews 8, e77–e84 (2007).

    Article  Google Scholar 

  11. Reddy, P., Najundaswamy, S., Mehta, R., Petrova, A. & Hegyi, T. Tin-mesoporphyrin in the treatment of severe hyperbilirubinemia in a very-low-birth-weight infant. J. Perinatol. 23, 507–508 (2003).

    Article  PubMed  Google Scholar 

  12. Dennery, P. A. Metalloporphyrins for the treatment of neonatal jaundice. Curr. Opin. Pediatr. 17, 167–169 (2005).

  13. Drummond, G. S. & Kappas, A. Chemoprevention of severe neonatal hyperbilirubinemia. Semin. Perinatol. 28, 365–368 (2004).

    Article  PubMed  Google Scholar 

  14. FDA Pediatric Advisory Committee. FDA Advisory Committee Briefing Document: Stannsoporfin for the Treatment of Neonatal Hyperbilirubinemia 1–162 (United States Food and Drug Administration (US-FDA), MD, USA, 2018).

  15. Kinnear, C., Moore, T. L., Rodriguez-Lorenzo, L., Rothen-Rutishauser, B. & Petri-Fink, A. Form follows function: nanoparticle shape and its implications for nanomedicine. Chem. Rev. 117, 11476–11521 (2017).

    Article  CAS  PubMed  Google Scholar 

  16. Pelaz, B. et al. Diverse applications of nanomedicine. ACS Nano 11, 2313–2381 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Walkey, C. D. & Chan, W. C. W. Understanding and controlling the interaction of nanomaterials with proteins in a physiological environment. Chem. Soc. Rev. 41, 2780–2799 (2012).

    Article  CAS  PubMed  Google Scholar 

  18. Wagner, V., Dullaart, A., Bock, A.-K. & Zweck, A. The emerging nanomedicine landscape. Nat. Biotechnol. 24, 1211–1217 (2006).

    Article  CAS  PubMed  Google Scholar 

  19. Doshi, N. & Mitragotri, S. Designer biomaterials for nanomedicine. Adv. Funct. Mater. 19, 3843–3854 (2009).

    Article  CAS  Google Scholar 

  20. de Lázaro, I. & Mooney, D. J. Obstacles and opportunities in a forward vision for cancer nanomedicine. Nat. Mater. 20, 1469–1479 (2021).

    Article  PubMed  CAS  Google Scholar 

  21. Barenholz, Y. C. In Handbook of Harnessing Biomaterials in Nanomedicine (ed. Peer, D.) 463–528 (Jenny Stanford Publishing, 2021).

  22. United States Food and Drug Administration (US-FDA). Drug Approval Package: Abraxane (Pcalitaxel Protein-Bound Particles) Injectable Suspension. Accessed 12 Dec 2021. https://www.accessdata.fda.gov/drugsatfda_docs/nda/2005/21660_AbraxaneTOC.cfm (2005).

  23. Adhikari, A., Polley, N., Darbar, S., Bagchi, D. & Pal, S. K. Citrate functionalized Mn3O4 in nanotherapy of hepatic fibrosis by oral administration. Future Sci. OA 2, FSO146 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Adhikari, A. et al. Redox nanomedicine ameliorates chronic kidney disease (CKD) by mitochondrial reconditioning in mice. Commun. Biol. 4, 1013 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Adhikari, A. et al. Incorporation of a biocompatible nanozyme in cellular antioxidant enzyme cascade reverses huntington’s like disorder in preclinical model. Adv. Healthc. Mater. 10, 2001736 (2021).

    Article  CAS  Google Scholar 

  26. Adhikari, A. et al. Manganese neurotoxicity: nano-oxide compensates for ion-damage in mammals. Biomater. Sci. 7, 4491–4502 (2019).

    Article  CAS  PubMed  Google Scholar 

  27. Xu, Z. et al. Facet-dependent biodegradable Mn3O4 nanoparticles for ameliorating Parkinson’s disease. Adv. Healthc. Mater. 10, 2101316 (2021).

    Article  CAS  Google Scholar 

  28. Aschner, J. L. & Aschner, M. Nutritional aspects of manganese homeostasis. Mol. Asp. Med. 26, 353–362 (2005).

    Article  CAS  Google Scholar 

  29. Avila, D. S., Puntel, R. L. & Aschner, M. In Metal Ions in Life Sciences Vol. 13 (eds Sigel, A., Sigel, H. & Sigel, R. K. O.) 199–227 (Springer Netherlands, 2013).

  30. Emsley, J. Manganese the protector. Nat. Chem. 5, 978–978 (2013).

    Article  CAS  PubMed  Google Scholar 

  31. Mattison, D. R. et al. Severity scoring of manganese health effects for categorical regression. NeuroToxicology 58, 203–216 (2017).

    Article  CAS  PubMed  Google Scholar 

  32. Li, L. & Yang, X. The essential element manganese, oxidative stress, and metabolic diseases: links and interactions. Oxid. Med. Cell. Longev. 2018, 7580707 (2018).

    PubMed  PubMed Central  Google Scholar 

  33. Mondal, S. et al. Novel one pot synthesis and spectroscopic characterization of a folate-Mn3O4 nanohybrid for potential photodynamic therapeutic application. RSC Adv. 9, 30216–30225 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Singh, N., Savanur, M. A., Srivastava, S., D’Silva, P. & Mugesh, G. A manganese oxide nanozyme prevents the oxidative damage of biomolecules without affecting the endogenous antioxidant system. Nanoscale 11, 3855–3863 (2019).

    Article  CAS  PubMed  Google Scholar 

  35. Yao, J. et al. ROS scavenging Mn3O4 nanozymes for in vivo anti-inflammation. Chem. Sci. 9, 2927–2933 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Huang, C.-C. et al. Chronic manganese intoxication. Arch. Neurol. 46, 1104–1106 (1989).

    Article  CAS  PubMed  Google Scholar 

  37. Crossgrove, J. & Zheng, W. Manganese toxicity upon overexposure. NMR Biomedicine 17, 544–553 (2004).

    Article  CAS  Google Scholar 

  38. Institute of Medicine (US) Panel on Micronutrients. Dietary Reference Intakes for Vitamin A, Vitamine K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc. 394–419 (Institute of Medicine (US) Washington DC, USA, 2001).

  39. Adhikari, A. et al. Redox buffering capacity of nanomaterials as an index of ROS-based therapeutics and toxicity: a preclinical animal study. ACS Biomater. Sci. Eng. 7, 2475–2484 (2021).

    Article  CAS  PubMed  Google Scholar 

  40. Giri, A. et al. Unprecedented catalytic activity of Mn3O4 nanoparticles: potential lead of a sustainable therapeutic agent for hyperbilirubinemia. RSC Adv. 4, 5075–5079 (2014).

    Article  CAS  Google Scholar 

  41. Rice, A. C. & Shapiro, S. M. A new animal model of hemolytic hyperbilirubinemia-induced bilirubin encephalopathy (kernicterus). Pediatr. Res. 64, 265–269 (2008).

    Article  CAS  PubMed  Google Scholar 

  42. Amini, N. et al. Animal kernicterus models: progress and challenges. Brain Res. 1770, 147624 (2021).

    Article  CAS  PubMed  Google Scholar 

  43. Bortolussi, G. & Muro, A. F. Experimental models assessing bilirubin neurotoxicity. Pediatr. Res. 87, 17–25 (2020).

    Article  PubMed  Google Scholar 

  44. Maity, S., Nag, N., Chatterjee, S., Adhikari, S. & Mazumder, S. Bilirubin clearance and antioxidant activities of ethanol extract of phyllanthus amarus root in phenylhydrazine-induced neonatal jaundice in mice. J. Physiol. Biochem. 69, 467–476 (2013).

    Article  CAS  PubMed  Google Scholar 

  45. Chen, S. & Tukey, R. H. Humanized UGT1 mice, regulation of UGT1A1, and the role of the intestinal tract in neonatal hyperbilirubinemia and breast milk-induced jaundice. Drug Metab. Disposition 46, 1745 (2018).

    Article  CAS  Google Scholar 

  46. Yueh, M.-F., Chen, S., Nguyen, N. & Tukey, R. H. Developmemntal onset of bilirubin-induced neurotoxicity involves toll-like receptor-2 dependent signalling in humanized UDP-glucuronosyltransferase1 mice. J. Biol. Chem. 289, 4699–4709 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Buege, J. A. & Aust, S. D. In Methods in Enzymology Vol. 52 (eds Fleischer, S. & Packer, L.) 302–310 (Academic Press, 1978).

  48. Adhikari, A. et al. A smart nanotherapeutic agent for in vitro and in vivo reversal of heavy-metal-induced causality: key information from optical spectroscopy. ChemMedChem 15, 420–429 (2020).

    Article  CAS  PubMed  Google Scholar 

  49. Benjamini, Y., Krieger, A. M. & Yekutieli, D. Adaptive linear step-up procedures that control the false discovery rate. Biometrika 93, 491–507 (2006).

    Article  Google Scholar 

  50. Adhikari, A., Mondal, S., Darbar, S. & Kumar Pal, S. Role of nanomedicine in redox mediated healing at molecular level. Biomolecular Concepts 10, 160–174 (2019).

    Article  CAS  PubMed  Google Scholar 

  51. Mondal, S. et al. Interaction of a jaundice marker molecule with a redox-modulatory nano-hybrid: a combined electrochemical and spectroscopic study toward the development of a theranostic tool. ChemMedChem e202100660 (2022).

  52. Morgan Chan, Z. et al. Electrochemical trapping of metastable Mn3+ ions for activation of MnO2 oxygen evolution catalysts. Proc. Natl Acad. Sci. USA 115, E5261–E5268 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  53. Polley, N. et al. Safe and symptomatic medicinal use of surface-functionalized Mn3O4 nanoparticles for hyperbilirubinemia treatment in mice. Nanomedicine 10, 2349–2363 (2015).

    Article  CAS  PubMed  Google Scholar 

  54. Organisation for Economic Co-operation and Development (OECD). Test No. 408: Repeated Dose 90-Day Oral Toxicity Study in Rodents (2018).

  55. Lei, S., Tang, K., Fang, Z. & Zheng, H. Ultrasonic-assisted synthesis of colloidal Mn3O4 nanoparticles at normal temperature and pressure. Cryst. Growth Des. 6, 1757–1760 (2006).

    Article  CAS  Google Scholar 

  56. Pike, J., Hanson, J., Zhang, L. & Chan, S.-W. Synthesis and redox behavior of nanocrystalline hausmannite (Mn3O4). Chem. Mater. 19, 5609–5616 (2007).

    Article  CAS  Google Scholar 

  57. Gagrani, A., Ding, B., Wang, Y. & Tsuzuki, T. pH dependent catalytic redox properties of Mn3O4 nanoparticles. Mater. Chem. Phys. 231, 41–47 (2019).

    Article  CAS  Google Scholar 

  58. Shapiro, S. M. Definition of the clinical spectrum of kernicterus and bilirubin-induced neurologic dysfunction (bind). J. Perinatol. 25, 54–59 (2005).

    Article  CAS  PubMed  Google Scholar 

  59. Cayabyab, R. & Ramanathan, R. High unbound bilirubin for age: a neurotoxin with major effects on the developing brain. Pediatr. Res. 85, 183–190 (2019).

    Article  CAS  PubMed  Google Scholar 

  60. Francis, R. O. et al. Glucose-6-phosphate dehydrogenase deficiency in transfusion medicine: the unknown risks. Vox Sanguinis 105, 271–282 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Funding

This study has been supported by Abdul Kalam Technology Innovation National Fellowship (INAE/121/AKF), Indian National Academy of Engineering (INAE), Science and Engineering Research Board (SERB), Department of Science and Technology (DST), Govt. of India and financial grant under BOOST scheme (339/WBBDC/1P-2/2013), Department of Biotechnology (DBT-WB), Govt. of West Bengal. M.D. received Junior Research Fellowship (JRF) from University Grants Commission (UGC), Govt. of India.

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Authors and Affiliations

  1. Department of Chemical, Biological and Macromolecular Sciences, S. N. Bose National Centre for Basic Sciences, Block JD, Sector 3, Salt Lake, Kolkata, 700106, India

    Aniruddha Adhikari, Susmita Mondal & Samir Kumar Pal

  2. Department of Neonatal and Developmental Medicine, Lucile Packard Children’s Hospital, Stanford University, 750 Welch Road, Palo Alto, CA, 94304, USA

    Vinod K. Bhutani

  3. Department of Zoology, Uluberia College, University of Calcutta, Uluberia, Howrah, 711315, India

    Monojit Das, Siddhartha Sankar Bhattacharya, Debasish Pal & Samir Kumar Pal

  4. Department of Zoology, Vidyasagar University, Rangamati, Midnapore, 721102, India

    Monojit Das

  5. Research and Development Division, Dey’s Medical Stores (Mfg.) Pvt. Ltd., 62 Bondel Road, Ballygunge, Kolkata, 700019, India

    Soumendra Darbar

  6. Technical Research Centre, S. N. Bose National Centre for Basic Sciences, Block JD, Sector 3, Salt Lake, Kolkata, 700106, India

    Ria Ghosh & Samir Kumar Pal

  7. Physical Chemistry – innoFSPEC, University of Potsdam, Am Mühlenberg 3, Golm, 14476, Potsdam, Germany

    Nabarun Polley

  8. Department of Pathology, Coochbehar Govt. Medical College and Hospital, Silver Jubilee Road, Coochbehar, 736101, India

    Anjan Kumar Das

  9. Department of Pediatric Medicine, Nil Ratan Sirkar Medical College and Hospital, 138 AJC Bose Road, Sealdah, Rajabazar, Kolkata, 700014, India

    Asim Kumar Mallick

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  1. Aniruddha Adhikari
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  12. Samir Kumar Pal
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Contributions

A.A., V.K.B., A.K.M and S.K.P. conceived and planned the study. A.A., S.M., M.D., N.P., and S.D. performed animal experiments and biochemical studies. A.A. executed numerical calculations and statistical analysis. A.A., S.D., S.S.B., D.P., A.K.M., V.K.B, and S.K.P. contributed to the interpretation of the results. A.K.D. carried out and evaluated the histological studies. A.A. and V.K.B. took the lead in writing the manuscript. All authors provided critical feedback and helped in shaping the research, analysis, interpretation, and writing. All authors reviewed, edited, and approved the final version of the manuscript.

Corresponding author

Correspondence to Samir Kumar Pal.

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Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

This study does not involve human subject or clinical study. Thus, patient consent was not required. All animal studies and experimental procedures were performed at Central Animal Facility, Department of Zoology, Uluberia College, India (Reg. No.: 2057/GO/ReRcBi/S/19/CPCSEA) following the protocol approved by the Institutional Animal Ethics Committee (Ref: 02/S/UC-IAEC/01/2019) as per standard guideline of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Govt. of India.

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Adhikari, A., Bhutani, V.K., Mondal, S. et al. Chemoprevention of bilirubin encephalopathy with a nanoceutical agent. Pediatr Res (2022). https://doi.org/10.1038/s41390-022-02179-5

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